DESCRIPTION (Adapted from applicant's Description): The elevation of intracellular free Ca2+ concentration is an essential signal controlling the differentiation and functions of T lymphocytes. The long-term goal of this proposal is to elucidate the molecular mechanisms responsible for generating the shaping Ca2+ signals in T Cells. Ca2+ signals in T cells are generated to a great extent by the activity of Ca2+ release-activated Ca2+ (CRAC) channels. These channels open in response to the depletion of intracellular Ca2+ stores, but the mechanism linking store depletion to channel opening is not well understood. We will test two possible mechanisms of CRAC channel activation using a combination of electrophysiological and fluorescence imaging approaches: regulated insertion of open channels into the plasma membrane by vesicle fusion, and control of CRAC channel gating by physical coupling to store membrane proteins. Mitochondria play an essential role in maintaining a high rate of Ca2+ influx through CRAC channels. To further understand how this function is carried out, we will examine the functional interactions between mitochondria and both CRAC channels and Ca2+-activated K+ channels, and relate this to the control of membrane potential and Ca2+ influx. Finally, Ca2+ ATPases in the plasma membrane (PMCA) are primarily responsible for the clearance of Ca2+ from T cells, and their activity is modulated slowly by changes in [Ca2+], allowing them to contribute to the complexity of Ca2+ signaling dynamics. We will apply a novel cytosolic calcium clamp technique to characterize the Ca2+- and time-dependence of PMCA modulation and its molecular mechanism. The significance of these studies is two-fold. First, CRAC channels, KCa channels, mitochondria, and pumps are widely expressed in various forms among non-excitable cells, so that a better understanding of their operation and interactions in T cells will shed light on Ca2+ signaling mechanisms in many cell types. Second, the complex nature of Ca2+ signals in T cells is known to be an important determinant for the selective regulation of downstream responses such as gene expression and cell activation during the immune response. Thus, the results of this study may help identify novel targets for the control of the immune response. Thus, the results of this study may help identify novel targets for the control of the immune response that may be beneficial in treating autoimmune disorders of immunodeficiencies, and they may help to explain immune dysfunctions resulting from the abberrant operation of channels, pumps and mitochondria.